The mismatch repair (MMR) pathway reduces mis-incorporation errors that occur during DNA replication. MMR proteins also prevent recombination between divergent DNA sequences, process recombination intermediates containing non-homologous ends during single strand annealing (SSA), and transduce DNA damage signals to cell cycle checkpoint and apoptosis pathways. The mechanisms by which these proteins recognize mismatches and other structures within recombination and replication intermediates and funnel them to downstream factors are not well understood; we are addressing these issues in S. cerevisiae through the following aims: 1. The effect of MMR and SGS1 helicase mutations will be examined in a new assay that analyzes homeologous recombination using both physical and genetic approaches. These studies should provide new insights into how recombination is regulated. 2. In yeast, the MSH2-MSH3 mismatch recognition complex and the RAD1-AD10 endonuclease play critical roles in removing nonhomologous 3' single strand tails during SSA. Because SSA is an important recombination pathway in both yeast and mammalian cells, we are developing an in vitro endonuclease assay to study interactions between these and other DNA repair factors. 3. Using a combination of chromatin-immunoprecipitation (CHIP), genetic, and physical assays, we are testing models aimed at understanding how the MSH2 MMR protein regulates recombination during mating type switching, a well defined recombination event. 4. We have identified mutations in the MLH1 MMR gene derived from the S288C yeast strain that disrupt MMR only when introduced into SK1 strains. The MMR defects observed in SK1 were eliminated when the S288C PMS1 MMR gene was co-introduced with the S288C mlh1 alleles. This result is encouraging us to test whether the MMR genes are co-evolving in S. cerevisiae. It also suggests how a MMR defective phenotype could arise in humans as the result of the segregation of naturally occurring polymorphisms.